MECHANISMS OF A1-ADRENOCEPTORS ON CARDIAC MYOCYTES

近年来，人们对 α肾上腺素能受体在调节 心脏功能和代谢。 本报告的总体目标 建议是以全面的方式定义分子 心脏α受体的作用机制。 研究将 使用分离的成人心室肌细胞进行。 的 工作将集中在阿尔法受体的两个已知结果， 被激动剂占据; i. 环磷酸腺苷降解活化 和二. 增加磷脂酰肌醇的水解。 HPLC- 检测将被用来充分表征的性质 由α受体刺激形成的糖磷酸盐， 它们在细胞中出现和消失的动力学。 稳定 GTP类似物将用于破碎细胞制剂， 研究GTP结合蛋白在激活 cAMP-磷酸二酯酶和磷脂酶C。 环状 AMP-磷酸二酯酶（PDE）活性将在 从肌细胞制备的亚细胞级分， 研究性质和细胞定位（胞质溶胶、SL、SR等） α-受体刺激的PDE。 SDS聚丙烯酰胺凝胶 电泳和等电聚焦将用于研究 假定的G蛋白的性质和它是一种 霍乱或百日咳毒素的底物。 Fura 2/AM 将采用肌细胞的标记来研究α-受体 调节心肌细胞中Ca++的释放。 这项工作将允许 磷酸肌醇的时间进程的仔细相关性 形成、Ca++释放和PDE激活。 α的调节 受体反应性将在用 佛波醇酯，以测试的假设，脱敏的 由于C-激酶依赖性磷酸化， G蛋白将α受体与其效应物偶联。 这些 研究将大大促进我们对 通过α受体调节心肌细胞代谢。 因为我们对cAMP的调节已经有了很多了解， 在这些单个肌肉细胞中的作用，很可能建议 关于α受体机制的工作可以详细解释 心肌细胞内发生的一系列事件， 内源性激动剂去甲肾上腺素刺激α和β 受体肯定发生在体内。 此外，这些研究 将提供一个基本框架，以评估阿尔法的变化 受体在疾病中的作用

During recent years there has been considerable interest in the role that alpha adrenergic receptors play in the regulation of cardiac function and metabolism. The overall objective of this proposal is to define in a comprehensive manner the molecular mechanism(s) of action of the cardiac alpha receptor. Studies will be conducted using the isolated adult ventricular myocyte. The work will focus on two known outcomes of alpha receptor occupation by agonist; i. activation of cyclic AMP degradation and ii. increased hydrolysis of phosphatidylinositol. HPLC- detection will be employed to fully characterize the nature of the sugar phosphates formed by alpha receptor stimulation and the kinetics of their appearance and disappearance in the cell. Stable GTP analogues will be used in broken cell preparations to investigate the role of a GTP-binding protein in the activation of both a cAMP- phosphodiesterase and phospholipase C. Cyclic AMP-phosphodiesterase (PDE) activity will be studied in subcellular fractions prepared from myocytes in order to investigate the nature and cellular location (cytosol, SL, SR etc.) of the alpha-receptor stimulated PDE. SDS polyacrylamide gel electrophoresis and isoelectric focusing will be used to study the nature of the putative G-protein and the possibility that it is a substrate for either cholera or pertussis toxins. Fura 2/AM labeling of myocytes will be employed to study the alpha-receptor regulation of Ca++ release in the myocyte. The work will permit a careful correlation of the time course of inositol phosphate formation, Ca++ release and PDE activation. Regulation of alpha receptor responsiveness will be studied in myocytes treated with phorbol esters to test the hypothesis that desensitization of response occurs due to C-kinase dependent phosphorylation of the G-protein coupling alpha receptors to their effector. These studies will significantly advance our understanding of the regulation of cardiac muscle cell metabolism by alpha receptors. Because much is already known about the regulation of cAMP action in these single muscle cells, it is likely that the proposed work on alpha receptor mechanisms can explain in detail the sequence of events occurring inside cardiac muscle cells when the endogenous agonist norepinephrine stimulates both alpha and beta receptors as surely occurs in vivo. Furthermore, these studies will provide a basic framework in which to assess changes in alpha receptor effects in disease.